CN116716201A - Enterococcus faecalis Y3 and probiotic preparation and application thereof - Google Patents
Enterococcus faecalis Y3 and probiotic preparation and application thereof Download PDFInfo
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- CN116716201A CN116716201A CN202211300737.6A CN202211300737A CN116716201A CN 116716201 A CN116716201 A CN 116716201A CN 202211300737 A CN202211300737 A CN 202211300737A CN 116716201 A CN116716201 A CN 116716201A
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- enterococcus faecalis
- faecalis
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an enterococcus faecalis (Enterococcus faecalis) Y3 for improving pig immunity, inhibiting bacteria, preventing or treating pig respiratory tract diseases and/or digestive tract diseases and/or preventing or treating swine fever diseases and a probiotic preparation containing the enterococcus faecalis (Enterococcus faecalis) Y3. The invention also discloses application of the enterococcus faecalis (Enterococcus faecalis) Y3 and probiotic preparation. The invention also discloses a medicament for improving immunity or inhibiting germs. The invention also discloses a method for improving the immunity of pigs, which comprises feeding pigs with the enterococcus faecalis (Enterococcus faecalis) Y3 and a probiotic preparation.
Description
Technical Field
The invention relates to a strain and application thereof, in particular to enterococcus faecalis (Enterococcus faecalis) Y3, a probiotic preparation and application thereof, and belongs to the field of animal husbandry.
Background
Under the cold condition in winter, the intensive cultivation has the contradiction point of environmental ventilation and heat preservation. In order to ensure the temperature of the pig house, ventilation is less. Because the raising density is too high, more dust exists in the pig house, and the higher dust density brings serious burden to the respiratory tract of pigs; meanwhile, pathogens exist in the dust, and the pig respiratory diseases are easy to occur; in addition, the bad ventilation causes the excessive ammonia concentration, stimulates the piglet respiratory mucosa, aggravates the occurrence of piglet respiratory diseases and secondary meningitis and digestive tract diseases. The piglets showed: elevated body temperature, inappetence or disability, mental depression, wheezing, cough, abdominal breathing, dyspnea, coarse and disordered hair, poor growth, and secretion from nose and eyes. At present, respiratory diseases in winter have become a main factor which plagues piglet cultivation.
At present, the main practice for preventing respiratory diseases is as follows: 1. and (5) enhancing the feeding management. The pigsty building structure needs to be warm in winter and cool in summer, meets the requirement of natural ventilation, and avoids the randomness of manual operation; according to the requirements of different pig raising stages, the raising density is reasonably arranged; and the manure is timely cleared, the ammonia content in the pig house is reduced, the damage to the respiratory tract mucous membrane of the pig is reduced, and the infection risk is reduced. 2. Improving immunity. Vaccine and veterinary drug prevention. The pathogens of the respiratory diseases are as follows: blue ear disease, infectious pleuropneumonia, swine plague, mycoplasma pneumonia, haemophilus parasuis, and infectious atrophic rhinitis. And reasonably injecting vaccine against pathogens. In addition, veterinary medicines and traditional Chinese medicines with preventive doses are added into feed and drinking water, so that the immunity of piglets is improved. However, frequent vaccine injections cause a large stress response, and the state advocates no-antibiotic cultivation, and reduces the preventive use of antibiotics, which limits the use of antibiotics in pig raising production.
Probiotics are a group of microorganisms beneficial to organisms, and through colonization in an animal body, the flora composition of a certain part of a host is changed. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced.
Probiotics have been developed to a certain extent in the fields of agriculture, medical treatment and health and industry, but at present, the probiotics in China are uneven in product, and have more defects. 1. The use of probiotic strains did not take into account the homology of the animals used. The probiotics for pigs are mainly obtained by separating plants, soil and heterologous animals; the strain is mostly led from abroad, such as bifidobacterium lactis is mostly used for treating respiratory diseases, but the bifidobacterium lactis is derived from human excrement and is often used in combination with other probiotics or traditional Chinese medicines, and the preparation steps are complicated (patent application numbers: 2012800285062, 2017104275011 and 2020100928167). 2. The evaluation of the probiotics characteristics is not enough, and whether the probiotics have the effects of improving the immune function, inhibiting pathogenic bacteria and the like or not is not clear, namely the probiotics are applied to clinic. 3. The activity of the strain and the number of viable bacteria are insufficient, so that the normal efficacy of the probiotics is difficult to ensure. 4. Probiotics are affected by the stock time, the number of living bacteria is rapidly reduced, and a protective agent is lacked. 5. The probiotics are improperly added when in use, and the effect of adding cannot be achieved.
In the prior art, cui Yuge et al (influence of compound probiotics and astragalus polysaccharide on the growth performance, immune function, serum biochemical index and diarrhea rate of weaned pigs, 2022), chloranthus (influence of probiotics on the intestinal flora and the growth performance of the pigs, 2021), chen Baojian et al (influence of adding compound probiotics into drinking water on the growth performance and the immune index of the pigs, 2020), liu Kewen (development and development of novel compound probiotics preparation for pigs, 2011) show that the probiotics can be immunized integrally, but do not mention whether the respiratory tract is protected.
Disclosure of Invention
The invention aims to: the technical problem to be solved by the invention is to provide the enterococcus faecalis (Enterococcus faecalis) Y3 for improving the immunity of pigs, inhibiting germs, preventing or treating diseases of the respiratory tract or/and the digestive tract of pigs and/or preventing or treating swine fever and a probiotic preparation containing the enterococcus faecalis (Enterococcus faecalis) Y3.
The invention also solves the technical problem of providing the application of the enterococcus faecalis (Enterococcus faecalis) Y3 and probiotic preparation.
The invention also aims to provide a medicine for improving immunity or inhibiting germs.
The technical problem to be solved by the invention is to provide a method for improving the immunity of pigs, which comprises feeding pigs with the enterococcus faecalis (Enterococcus faecalis) Y3 and a probiotic preparation.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a enterococcus faecalis (Enterococcus faecalis) Y3, wherein the enterococcus faecalis (Enterococcus faecalis) Y3 is preserved in China general microbiological culture collection center (CGMCC) for culture Collection of microorganisms in the year 2022, month 6 and day 10, and the preservation address is as follows: beijing, china, with the preservation number of CGMCC NO.25043.
The invention also provides a probiotic preparation containing the enterococcus faecalis (Enterococcus faecalis) Y3.
Wherein the probiotic preparation comprises one or more of liquid preparation, powder, capsule and tablet.
The invention also provides application of the enterococcus faecalis (Enterococcus faecalis) Y3 or probiotic preparation in preparation of medicines for improving immunity and/or preventing or treating diseases of the respiratory tract and/or the digestive tract of pigs and/or preventing or treating swine fever.
The invention also provides application of the enterococcus faecalis (Enterococcus faecalis) Y3 or probiotic preparation in preparing medicaments for inhibiting bacteria.
Wherein the bacteria comprise one or more of escherichia coli, salmonella or staphylococcus.
The invention also provides a medicament for improving immunity, which comprises the enterococcus faecalis (Enterococcus faecalis) Y3 or the probiotic preparation.
The invention also provides a medicine for inhibiting bacteria, which comprises the enterococcus faecalis (Enterococcus faecalis) Y3 or the probiotic preparation.
Wherein the bacteria comprise one or more of escherichia coli, salmonella or staphylococcus.
The invention also provides a method of improving the immunity of a pig, the method comprising feeding the pig with the enterococcus faecalis (Enterococcus faecalis) Y3 or probiotic preparation.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
1. the invention obtains the enterococcus faecalis (Enterococcus faecalis) Y3 which can improve the immunity of pigs, inhibit germs, prevent or treat diseases of the respiratory tract and/or the digestive tract of the pigs and/or prevent or treat swine fever for the first time. The method combines animal nutrition from the aspect of microorganism specialty, develops homologous targeted screening and directional feeding, overcomes the defect that the sources of strains are led from abroad, breaks through the technical barriers, autonomously develops functional probiotic preparations, ensures the number of live bacteria of the probiotics and the number of live bacteria of storage shelves, adopts a reasonable adding mode, is directly added in the drinking water of weaned pigs, and is convenient to operate. The invention is applied to pig raising production of piglets in winter, improves the immunity and disease resistance of the piglets and relieves the difficult problem of respiratory diseases. Meanwhile, after the probiotics are added to the nursery pig for 28 days, the production performance of the nursery pig can be obviously improved. The probiotics E.faecalis Y3 liquid preparation is superior to other similar products in serving as a green pollution-free microecological preparation, and can be applied to the production of piglets in winter.
2. The probiotic preparation developed by the invention is separated from Meishan pigs, is a high-quality black pig variety in Jiangsu places, has the feeding habit similar to that of wild pigs, and has more abundant intestinal flora. After the obtained probiotics strain is developed into a liquid preparation, and then the liquid preparation is added to pigs, so that the homology is met. Meanwhile, the number of the viable bacteria reaches 10 percent of the viable bacteria of the feed microorganism in China 6 CFU/g. The shelf time reaches 90 days, and the number of viable bacteria still reaches 10 9 CFU/g. The quantity of viable bacteria is slightly affected by the storage time. The preparation of the invention is added into drinking water, and after the nursery pig drinks for 28 days through the alimentary canal, the production performance of the nursery pig can be obviously improved. Effects on immune function: not only improves the blood immunoglobulin G, M, A and swine fever antibody level, but also improves the expression level of pulmonary mucosa immune factors IFN-a, beta-PBD 1 and TNF-a and IgA protein level, and plays a role in protecting respiratory tract.
In conclusion, the E.faecalis Y3 is prepared into a liquid preparation, and can be applied to pig raising production of piglets in winter, so that the problems of improving the growth performance, improving the immunity and disease resistance of the piglets and relieving respiratory diseases in winter can be solved.
Drawings
FIG. 1 shows the final body weights of different groups of nursery pigs;
FIG. 2 shows average daily gain of nursery pigs of different groups;
FIG. 3 shows the feed to meat ratio of different groups of nursery pigs;
FIG. 4 shows the lung tissue immune factor mRNA expression level (IFN-a) of different groups of nursery pigs;
FIG. 5 shows the lung tissue immune factor mRNA expression levels (. Beta. -PBD 1) of different groups of nursery pigs;
FIG. 6 shows the levels of immune factor mRNA expression (TNF-a) in lung tissue of various groups of nursery pigs;
FIG. 7 shows the respiratory and digestive tract disease incidence and mortality of different groups of nursery pigs.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The invention separates a plurality of probiotics from the intestinal tract of a healthy Jiangsu local pig (Meishan pig), and then obtains 1 strain of probiotics bacillus through digestive enzyme production evaluation, gastric acid resistance, bile salt resistance and thermal environment evaluation, which is enterococcus faecalis Y3 (Enterococcus faecalis, E.faecalis) Y3, and is preserved in the common microorganism center of China microbiological culture Collection center at the 6 th month 10 of 2022, wherein the preservation address is Beijing China, the preservation number is CGMCC NO.25043, and the classification name is enterococcus faecalis (Enterococcus faecalis) which is derived from pigs.
The pathogenic bacteria indicator bacteria are respectively: coli (accession No. cctccc 25922) was purchased from the China general microbiological culture collection center, salmonella of swine origin (accession No. CVCC1880, salmonella) and staphylococcus aureus (accession No. CVCC1882, staphylococcus), respectively, from the China veterinary microbiological culture collection center.
Liquid nutrient agar culture medium is prepared, enterococcus faecalis Y3, escherichia coli, salmonella suis and staphylococcus aureus are respectively inoculated into the liquid nutrient agar culture medium (containing beef extract 3g, peptone 10g, sodium chloride 5g and agar 3g in 1000 ml of water) for activation culture for 16 hours for later use.
The enterococcus faecalis Y3 is co-cultured with escherichia coli CCTCC25922, salmonella CVCC1880 of swine origin and staphylococcus aureus CVCC1882 respectively. Respectively taking 0.2mL of escherichia coli CCTCC25922, swine salmonella CVCC1880 and staphylococcus aureus CVCC1882 to 5mL of liquid nutrient agar culture medium, and then respectively inoculating 0.2mL of pig enterococcus faecalis Y3; viable counts were made at 0h, 12h, 24h, 36h, respectively. Meanwhile, escherichia coli CCTCC25922, salmonella suis CVCC1880 and Staphylococcus aureus CVCC1882 were cultured separately in nutrient agar medium (1000 ml: beef extract 3g, peptone 10g, sodium chloride 5g, agar 3 g) and counted as controls. Comparing the number of viable bacteria of the mixed culture group and the number of viable bacteria of the 3 pathogenic bacteria control group, and judging whether the pig enterococcus faecalis (E.faecalis) Y3 has the capability of inhibiting the escherichia coli or not.
TABLE 1
And (3) table notes: the same row of shoulder marks with different capital letters indicates that the difference is very significant (P < 0.01), the following is the same.
The results are shown in Table 1, and after the enterococcus faecalis (E.faecalis) Y3 and the salmonella CVCC1880 are co-cultured for 12 hours, the number of viable salmonella CVCC1880 is reduced to 4.51X10 4 After CFU/mL and 24 hours, the number of salmonella CVCC1880 viable bacteria in the culture solution is reduced to 0CFU/mL, no salmonella is detected after 36 hours, and the number is extremely obviously lower than that of a control group (P)<0.01). After E.faecalis Y3 and staphylococcus aureus CVCC1882 are co-cultured for 12 hours, the number of viable staphylococcus aureus CVCC1882 is reduced to 5.63 multiplied by 10 4 After the culture medium is subjected to CFU/mL for 24 hours, the number of the staphylococcus aureus CVCC1882 viable bacteria in the culture medium is reduced to 0CFU/mL, and the staphylococcus aureus CVCC1882 is not detected for 36 hours. E.faecalis Y3 and Escherichia coli CCTCC25922 were co-cultured for 12 hours, wherein the number of Escherichia coli CCTCC25922 was 1×10 from the control group 9 CFU/mL was reduced to 1.82×10 3 After the culture solution reaches CFU/mL and 24 hours, the number of the escherichia coli CCTCC25922 in the culture solution is reduced to 0CFU/mL, and the number of the escherichia coli cannot be detected in the culture solution for 36 hours.
Example 2 preparation of liquid probiotic formulations
1) Bacterial strain
Enterococcus faecalis Y3 (Enterococcus faecalis, E.faecium Y3) preserved by the China general microbiological culture Collection center is selected, the preservation date is 2022, 6 and 10 days, and the preservation number is CGMCC NO.25043.
2) Activation of bacterial species and obtaining of seed solution
E.faecalis Y3 stored at 4 ℃ is inoculated on MRS liquid culture medium according to an inoculum size of 1%, cultured for 12 hours at 37 ℃, and continuously activated for 2 times, so as to obtain bacterial seed liquid.
3) Production of
E.faecalis Y3 seed solution was inoculated in a specific medium at an inoculum size of 1%. The specific culture medium comprises the following components in percentage: feed grade molasses 8%, urea 5And (3) stirring and uniformly mixing the raw sugar 1%, placing the mixture in a 100L fermentation tank for high-pressure sterilization, and cooling to room temperature. Then inoculating E.faecium Y3 according to the volume ratio of 2%, culturing for 18h, and collecting liquid bacterial liquid, namely the probiotic preparation, wherein the number of viable bacteria is 4.2+/-0.3 multiplied by 10 9 CFU/g. Placing in a refrigerator at 4 ℃ for standby.
4) Viable count of product
Detecting the viable bacteria quantity of E.faecalis Y3 in the fermentation liquor by using a selective counting culture medium and adopting a plate counting method to reach 8.5x10 9 Viable bacteria units per milliliter (CFU/mL).
5) Shelf time
And (3) storing the sealed probiotic preparation at room temperature, and detecting the number of E.faecalis Y3 viable bacteria in the probiotic preparation according to the previous method after 30 days, 60 days and 90 days respectively. The results show that: the viable count after 30 days is 2.3+/-0.2 multiplied by 10 9 CFU/g, viable count after 60 days is 1.6+ -0.2X10 9 CFU/g, viable count after 90 days is 1.4+ -0.3X10 9 CFU/g. The quantity of the microbial ecological additive exceeds the content requirement of the living bacteria of the national feeding microecological additive>10 8 CFU/g)。
Example 3 probiotic formulations improve the immunity and growth of nursery pigs
1) Test animals and groups
180 weaned pigs (Du long commercial pigs) with a weight of about 6.8kg are selected and randomly divided into 3 groups, namely a control group, an E.faecalis Y3 additive group and a drug group. Each group was fed with a corresponding diet for 4 replicates of 15 piglets each. The daily ration is as follows: basal ration (nutrition level reaches NRC standard of weaned pigs); e.faecalis Y3 additive amount group, 2.5L E.faecalis Y3 liquid fermentation product is added into each ton of drinking water, and the number of viable bacteria is 1.0+/-0.2 multiplied by 10 7 CFU/g. Free drinking water; drug group: 400g of flavomycin is added into each ton of feed and is uniformly mixed for standby. The experimental nursery pigs are fed and immunized normally. The period was 35 days. The basic ration formulation is shown in table 2 below.
TABLE 2
Note that: vitamins and minerals per kg ration: v (V) D3 13000IU，V E 200IU，V K 3 20mg，V B1 30mg，V B2 50mg，V B6 50mg，V B12 0.2mg，V C 600mg, 100mg of manganese, 80mg of zinc, 80mg of iron, 10mg of copper, 0.15mg of iodine, 0.2mg of selenium and 0.2mg of chromium.
2) Feeding management
Each group is independently fed with the feed correspondingly prepared, and the drinking trough is sterilized every day: once in the morning and afternoon. The feed is added for 4 times a day: each of the two times of morning and afternoon.
The weight of each group of feeds was recorded, the amount of feed remaining in each group was weighed by a scale after the test was completed, the weight of each group of nursery pigs was weighed after 35 days, and the growth performance data was compared. The number of respiratory tract disease attacks per day and the number of other disease attacks per group and the treatment cost were recorded.
3) Growth performance
The results of the growth performance of the three groups of piglets after 35 days of the test are shown in table 3.
TABLE 3 Table 3
Note that: * the difference is significant, (P<0.05)。
As shown in Table 3 and FIG. 1, the final weight of the E.faecalis Y3 group of nursery pigs was 19.65kg, the average weight of the drug group of nursery pigs was 19.62kg, and the average weight of the non-resistant control group of nursery pigs was 18.01kg. The body weight of the nursery pigs in the E.faecalis Y3 group and the drug group is obviously higher than that in the control group (P < 0.05), and no obvious difference exists between the E.faecalis Y3 group and the drug group (P > 0.05).
As shown in table 3 and fig. 2, the average daily gain of the control, e.faecalis Y3 and drug groups was 318g, 361g, 360g, respectively, and it was found that the e.faecalis Y3 and drug groups were significantly higher than the control group (P < 0.05), while there was no significant difference (P > 0.05) between the e.faecalis Y3 and drug groups.
Average daily feed intake: there was no significant difference between the three groups (P > 0.05).
As shown in table 3 and fig. 3, the meat proportions of the control group, e.faecalis Y3 group and drug group were 1.45, 1.30, 1.31, respectively. Both the faecalis Y3 group and the drug group were significantly lower than the control group (P < 0.05), while there was no significant difference between the e.faecalis Y3 group and the drug group (P > 0.05).
Example 4 Effect of probiotics on porcine pulmonary immune factor RNA levels
1. Total RNA extraction from samples
Collecting three groups (control group, E.faecalis Y3 group and drug group) of pig lung samples, shearing about 50mg of tissue samples in a freezing tube by using a sterilizing scissors, putting the frozen tissue samples in a 2mL centrifuge tube without RNase, adding 2 steel balls, adding 1000 mu L of tissue lysate (Trizol, simer flying company, product number 15596026), covering a cover, putting the tissue lysate in a high-channel tissue homogenizer, running the tissue homogenizer for 180s at 40Hz, taking out the centrifuge tube, sucking homogenate by using a 1mL suction head without RNase, centrifuging the homogenate in a new 1.5mL centrifuge tube without RNase for 2min at 4 ℃, sucking supernatant in the new 1.5mL centrifuge tube without RNase, carrying out a total RNA extraction experiment, and the extraction method is referred to the molecular biology laboratory manual.
2. Reverse transcription System and procedure
Reverse transcription system and conditions: 20. Mu.L reverse transcription system: 2 xRT Buffer 10. Mu.L, RT/RI 1. Mu.L, gDNA Remove 1. Mu.L, random Primer (10. Mu. Mol/L) 1. Mu.L, RNA 1.5. Mu.L (about 600 ng), RNase free dH 2 O7.5. Mu.L. Mixing the components uniformly, placing in a water bath kettle at 42 ℃ for incubation for 1H to obtain first strand cDNA, and using H to carry out reverse transcription on the cDNA 2 After O5-fold dilution, a subsequent qPCR reaction was performed.
3. Real-Time PCR reaction system and step
Performing mRNA expression quantity analysis of interferon a (IFN-a), defensin beta (beta-PBD 1) and tumor necrosis factor a (TNF-a) by adopting SYBR Green I method real-time fluorescence quantitative PCR (relative quantification); the primers for IFN-a, β -PBD1 and TNF-a are shown in Table 4 below, and the 20. Mu.L reaction scheme is optimized as follows: 2 xTop Green EX-Taq Mix 10. Mu.L, primer F (10. Mu. Mol/L) 0.5. Mu.L, primer R (10. Mu. Mol/L) 0.5. Mu.L, template cDNA 2. Mu.L, RNase Free dH 2 O 7μL。
TABLE 4 Table 4
By 2 -△△Ct The method is used for calculating the relative expression quantity.
4. Immune factor RNA levels
mRNA gene expression of IFN-a, beta-PBD 1 and TNF-a in pig lung tissue 35 days after feeding the nursery pigs with probiotics is shown in FIGS. 4-6. TNF-a: the TNF-a gene level of the lung tissue of the fasciasis Y3 added group is extremely higher than that of a control group and a drug group (P < 0.01), and the drug group is extremely higher than that of the control group (P < 0.01); beta-PBD 1: the faecalis Y3 group is extremely higher than the control group and the drug group (P < 0.01), and the difference between the drug group and the control group is not obvious (P > 0.05); IFN-a: the faecalis Y3 group was significantly higher than the control and drug groups (P < 0.01), and the difference between the drug and control groups was not significant (P > 0.05).
5. Lung and blood immune index of nursery pig
At 21 days of age in three groups of nursery pigs, swine fever vaccine was intramuscular injected, pig blood was collected for 28 days, and IgG, igM and swine fever antibody levels and lung tissue secretory IgA levels in the blood were detected. Pig immunoglobulin G, M, A (IgG, igM, igA) enzyme-linked immunosorbent kit, from Nanjing established biosystems, swine fever antibody kit, from Edison IDEXX, U.S.A. The results of the immune index are shown in table 5 35 days after feeding the probiotics.
TABLE 5
The lung tissue secretion type IgA level of the nursery pig in the E.faecalis Y3 group is extremely higher than that of the control group and the drug group (P < 0.01), and no obvious difference exists between the drug group and the control group (P > 0.05).
After 21 days of immunization of piglets with swine fever vaccine, blood is collected for 28 days to detect swine fever antibody titer, and the result shows that: the swine fever antibody level in the blood of the nursery pigs of the faecalis Y3 group is extremely higher than that of the control group and the drug group (P < 0.01), and the drug group is extremely higher than that of the control group (P < 0.01). The IgM and IgG levels in the blood of the nursery pigs of the faecalis Y3 group are extremely higher than those of the control group and the drug group (P < 0.01), and the IgG and IgM levels in the blood of the piglets of the drug group are not significantly different from those of the control group (P > 0.05).
6. Respiratory tract and digestive tract diseases and death rate of nursery pigs
Three groups of nursery pigs were observed daily for the number of coughs, diarrhea episodes and total number of medications, and total mortality was recorded over the 28 days of the trial. The respiratory tract, digestive tract and death rate of the nursery pigs are shown in figure 7. Incidence of respiratory disease: the nursery pigs in the E.faecalis Y3 group are obviously lower than those in the control group (P < 0.01), the difference between the nursery pigs in the E.faecalis Y3 group and the drug group is not obvious (P > 0.05), and the control group, the E.faecalis Y3 group and the drug group are 28.89%, 6.67% and 7.89% respectively. Incidence of digestive tract diseases: the nursery pigs in the E.faecalis Y3 group are obviously lower than those in the control group (P < 0.01), the difference between the nursery pigs in the E.faecalis Y3 group and the drug group is not obvious (P > 0.05), and the control group, the E.faecalis Y3 group and the drug group are respectively 13.67%, 4.76% and 5.12%. Rate of death: the nursery pigs in the E.faecalis Y3 group are obviously lower than those in the control group and the drug group (P < 0.01), and the control group, the E.faecalis Y3 group and the drug group are respectively 8.71%, 3.21% and 4.78%.
Claims (10)
1. Enterococcus faecalis strainEnterococcus faecalis) Y3 is characterized in that the enterococcus faecalis is prepared from the following components in percentage by weightEnterococcus faecalis) Y3, the preservation date is 2022, 6 and 10, and is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC NO.25043.
2. A probiotic formulation comprising enterococcus faecalis (Enterococcus faecalis) Y3 according to claim 1.
3. The probiotic preparation according to claim 2, characterized in that the probiotic preparation comprises one or several of liquid preparation, powder, capsule, tablet.
4. Use of enterococcus faecalis (Enterococcus faecalis) Y3 according to claim 1 or a probiotic formulation according to claim 2 for the manufacture of a medicament for improving immunity or for the prevention or/and treatment of a porcine respiratory disease or/and a digestive disease and/or a swine fever disease.
5. Use of enterococcus faecalis (Enterococcus faecalis) Y3 according to claim 1 or a probiotic preparation according to claim 2 for the manufacture of a medicament for inhibiting a pathogen.
6. The use according to claim 5, wherein the pathogen comprises one or more of escherichia coli, salmonella or staphylococcus.
7. An immunity enhancing medicament comprising enterococcus faecalis (Enterococcus faecalis) Y3 according to claim 1 or a probiotic formulation according to claim 2.
8. A medicament for inhibiting pathogens comprising enterococcus faecalis (Enterococcus faecalis) Y3 according to claim 1 or the probiotic formulation according to claim 2.
9. The medicament according to claim 8, wherein the pathogens comprise one or more of escherichia coli, salmonella or staphylococcus.
10. A method of increasing swine immunity comprising feeding swine with the enterococcus faecalis (Enterococcus faecalis) Y3 of claim 1 or the probiotic formulation of claim 2.
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